Store-operated calcium entry (SOCE) constitutes the primary calcium influx pathway in cells of the immune system. Dysregulated Ca2+ influx is intimately involved in primary immunodeficiency, cardiovascular remodeling, and tumor metastasis. SOCE occurs when STIM1, the calcium sensor in the endoplasmic reticulum (ER), senses depletion of ER calcium stores; in response, activated STIM1 migrates toward ER-plasma membrane (PM) junctions, where it recruits and gates the PM calcium channels ORAI (ORAI1, ORAI2 and ORAI3). Dynamic STIM-ORAI coupling represents a totally new paradigm for channel activation, and is currently being targeted for treatment of immuno- inflammatory diseases (e.g., plaque psoriasis). Critical barriers in our progress to understanding this important physiological process include: (i) how the store depletion signal is transmitted from the ER lumen to the cytoplasm; (ii) how STIM1 differentially couples to ORAI1 and ORAI3, the two major ORAI proteins that respond differently to pharmacological stimuli and cause distinct signaling phenotypes; and (iii) how ER-PM junctions dedicated to calcium influx are generated by hitherto uncharacterized regulators. The overall goal of this proposal is to tackle these unmet challenges. In Aim 1, we will use biochemical, protein engineering, and chemical biology approaches to establish the irreplaceable role of the STIM1 transmembrane domain in signal transduction. Our preliminary studies have suggested that the often-neglected single transmembrane domain may serve as the key determinant in relaying signals across the ER membrane and contribute to conformational switch in the cytoplasmic side of STIM1. In Aim 2, we will provide the first structural comparison between STIM1-ORAI1 and STIM1-ORAI3 coupling at atomic resolution. A model system for quantitative dissection of SOCE at the inter-membrane interface and a new engineered optogenetic tool for noninvasive control of puncta formation and calcium flux will be devised and used to aid structure-function studies and to gain stoichiometric and regulatory information on STIM1-ORAI coupling. The emerging significance of ER-PM junctions has recently received high attention. However, mechanistic dissection of this specialized cellular compartment is greatly hampered by the lack of appropriate tools and methods. In Aim 3, we will overcome this barrier by taking a two-pronged approach: (i) proteomic mapping of intact ER-PM junctions, which is made possible through spatially restricted in situ protein labeling, and (ii) screening based on bimolecular fluorescence complementation. Our pilot study using this strategy has already unveiled previously unrecognized STIM1 binding partner proteins at puncta. We will further expand this to identify additional novel regulators and generate corresponding cell lines through genome editing, which will be used to define the roles of those regulators in modulating SOCE, puncta formation, ER morphology, and T cell activation. Taken together, we expect that the novel mechanistic and structural insights gained through our study will lead to advances in effective treatment of autoimmune diseases and prevention of transplant rejection. Further benefit will accrue to other research areas that involve calcium signaling and intermembrane communication.